Mutable Collagenous Tissue Isolated from Echinoderms Leads to the Production of a Dermal Template That Is Biocompatible and Effective for Wound Healing in Rats.

The mutable collagenous tissue (MCT) of echinoderms possesses biological peculiarities that facilitate native collagen extraction and employment for biomedical applications such as regenerative purposes for the treatment of skin wounds. Strategies for skin regeneration have been developed and dermal substitutes have been used to cover the lesion to facilitate cell proliferation, although very little is known about the application of novel matrix obtained from marine collagen. From food waste we isolated eco-friendly collagen, naturally enriched with glycosaminoglycans, to produce an innovative marine-derived biomaterial assembled as a novel bi-layered skin substitute (Marine Collagen Dermal Template or MCDT). The present work carried out a preliminary experimental in vivo comparative analysis between the MCDT and Integra, one of the most widely used dermal templates for wound management, in a rat model of full-thickness skin wounds. Clinical, histological, and molecular evaluations showed that the MCDT might be a valuable tool in promoting and supporting skin wound healing: it is biocompatible, as no adverse reactions were observed, along with stimulating angiogenesis and the deposition of mature collagen. Therefore, the two dermal templates used in this study displayed similar biocompatibility and outcome with focus on full-thickness skin wounds, although a peculiar cellular behavior involving the angiogenesis process was observed for the MCDT.

A Second Life for Seafood Waste: Therapeutical Promises of Polyhydroxynapthoquinones Extracted from Sea Urchin by-Products.

Coping with a zero-waste, more sustainable economy represents the biggest challenge for food market nowadays. We have previously demonstrated that by applying smart multidisciplinary waste management strategies to purple sea urchin (Paracentrotus lividus) food waste, it is possible to obtain both a high biocompatible collagen to produce novel skin substitutes and potent antioxidant pigments, namely polyhydroxynapthoquinones (PHNQs). Herein, we have analyzed the biological activities of the PHNQs extract, composed of Spinochrome A and B, on human skin fibroblast cells to explore their future applicability in the treatment of non-healing skin wounds with the objective of overcoming the excessive oxidative stress that hinders wound tissue regeneration. Our results clearly demonstrate that the antioxidant activity of PHNQs is not restricted to their ability to scavenge reactive oxygen species; rather, it can be traced back to an upregulating effect on the expression of superoxide dismutase 1, one of the major components of the endogenous antioxidant enzymes defense system. In addition, the PHNQs extract, in combination with Antimycin A, displayed a synergistic pro-apoptotic effect, envisaging its possible employment against chemoresistance in cancer treatments. Overall, this study highlights the validity of a zero-waste approach in the seafood chain to obtain high-value products, which, in turn, may be exploited for different biomedical applications.

Assessment of the quality of the healing process in experimentally induced skin lesions treated with autologous platelet concentrate associated or unassociated with allogeneic mesenchymal stem cells: preliminary results in a large animal model.

Regenerative medicine for the treatment of skin lesions is an innovative and rapidly developing field that aims to promote wound healing and restore the skin to its original condition before injury. Over the years, different topical treatments have been evaluated to improve skin wound healing and, among them, mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) have shown promising results for this purpose. This study sought to evaluate the quality of the healing process in experimentally induced full-thickness skin lesions treated with PRP associated or unassociated with MSCs in a sheep second intention wound healing model. After having surgically created full-thickness wounds on the back of three sheep, the wound healing process was assessed by performing clinical evaluations, histopathological examinations, and molecular analysis. Treated wounds showed a reduction of inflammation and contraction along with an increased re-epithelialization rate and better maturation of the granulation tissue compared to untreated lesions. In particular, the combined treatment regulated the expression of collagen types I and III resulting in a proper resolution of the granulation tissue contrary to what was observed in untreated wounds; moreover, it led to a better maturation and organization of skin adnexa and collagen fibers in the repaired skin compared to untreated and PRP-treated wounds. Overall, both treatments improved the wound healing process compared to untreated wounds. Wounds treated with PRP and MSCs showed a healing progression that qualitatively resembles a restitutio ad integrum of the repaired skin, showing features typical of a mature healthy dermis.

Case report: Flexor carpi ulnaris tendinopathy in a lure-coursing dog treated with three platelet-rich plasma and platelet lysate injections.

In the present case report a 7-year-old male Whippet competing in lure-coursing presented with third-degree recurrent lameness of the right forelimb, pain on palpation of the caudal aspect of the carpus and swelling of the forearm proximally to the accessory carpal bone. Clinical, radiographic, and ultrasonographic evaluation diagnosed a flexor carpi ulnaris (FCU) chronic tendinopathy unresponsive to previously attempted conservative treatments such as oral non-steroidal anti-inflammatory drugs (NSAIDs) administration along with padded palmar splint application and rest. The dog was subjected to one injection of autologous platelet-rich plasma (PRP) obtained using a double centrifugation tube method, followed by two platelet lysate (PL) injections. Treatment was administered at three-week intervals. The healing process was assessed through clinical and ultrasonographic imaging (US) on the day of the first injection (T0), and at week three (T1), six (T2), twelve (T3), fifty-two (T4), and one-hundred-and-four (T5). Fiber alignment score (FAS) and echogenicity score (ES) were developed by modifying a previously published US assessment scale. At T1, ES, and FAS improvement was detected, and at T2, further improvements in ES and FAS were observed. Ultrasonographic results were clinically consistent with the improvement in lameness: lameness grade 3/4 was detected at T0 and grade 2/4 at T1. A lameness grade of 1/4 was detected at T2, and grade 0/4 was observed at T3, T4, and T5. Moreover, at T5, the dog returned to competition, and no history of re-injury was reported. Our results suggest that the treatment of FCU tendinopathy in lure-coursing dogs with a combination of consecutive injections of autologous PRP and PL could be feasible. Additionally, no adverse reactions were observed.

Case Report: Repeated Intralesional Injections of Autologous Mesenchymal Stem Cells Combined With Platelet-Rich Plasma for Superficial Digital Flexor Tendon Healing in a Show Jumping Horse.

In the present case report a show jumping 10-year-old Sella Italiano gelding, presented with severe lameness, swelling and pain at palpation of the mid-metacarpal region of the left forelimb. Clinical and ultrasound examination diagnosed a chronic tendonitis of the central region of the superficial digital flexor tendon (SDFT). The lesion was a reoccurrence since it developed from a previously healed injury. The horse had to stop competing and was unresponsive to gold-standard treatments as Non-steroidal anti-inflammatory drugs (NSAIDs) and conservative management after 6 months of therapy. The animal was subjected to repeated intralesional injections of autologous adipose-derived mesenchymal stem cells (AD-MSCs) combined with autologous platelet-rich plasma (PRP). The combined treatment was administered twice in a 1-month interval. The healing process was assessed through clinical examination, ultrasound imaging and quantification of oxidative stress products and inflammatory mediators in blood plasma. After 2 weeks from first injection, a reduction of concentration of oxidative-derived products was observed, together with an increase of anti-inflammatory cytokines and pro-mitotic growth factors. These results were reflected clinically as the horse showed a reduction of lameness along with swelling and pain after 4 weeks. At the 1-year follow-up, the horse showed no signs of lameness and swelling. The ultrasonographic examination highlighted a compact fiber alignment with a normal echogenic tendon as observed in the sound contralateral limb. Moreover, the horse went back to the previous level of competition. Our results suggest the positive effects of a repeated intralesional injection of AD-MSCs and PRP for the treatment of a chronic tendonitis with long-term effects and an improvement for both equine quality of life and athletic performance.

A Prototype Skin Substitute, Made of Recycled Marine Collagen, Improves the Skin Regeneration of Sheep.

Skin wound healing is a complex and dynamic process that aims to restore lesioned tissues. Collagen-based skin substitutes are a promising treatment to promote wound healing by mimicking the native skin structure. Recently, collagen from marine organisms has gained interest as a source for producing biomaterials for skin regenerative strategies. This preliminary study aimed to describe the application of a collagen-based skin-like scaffold (CBSS), manufactured with collagen extracted from sea urchin food waste, to treat experimental skin wounds in a large animal. The wound-healing process was assessed over different time points by the means of clinical, histopathological, and molecular analysis. The CBSS treatment improved wound re-epithelialization along with cell proliferation, gene expression of growth factors (VEGF-A), and development of skin adnexa throughout the healing process. Furthermore, it regulated the gene expression of collagen type I and III, thus enhancing the maturation of the granulation tissue into a mature dermis without any signs of scarring as observed in untreated wounds. The observed results (reduced inflammation, better re-epithelialization, proper development of mature dermis and skin adnexa) suggest that sea urchin-derived CBSS is a promising biomaterial for skin wound healing in a "blue biotechnologies" perspective for animals of Veterinary interest.

Could cold plasma act synergistically with allogeneic mesenchymal stem cells to improve wound skin regeneration in a large size animal model?

Skin wound healing may sometimes lead to open sores that persist for long periods and expensive hospitalization is needed. Among different kinds of therapeutic innovative approaches, mesenchymal stem cells (MSCs) and low-temperature atmospheric pressure cold plasma (ionized gas) have been recently tested to improve this regenerative process. To optimize wound healing the present study intended to combine, for the first time, these two novel approaches in a large size animal wound healing model with the aim of assessing the putative dual beneficial effects. Based on clinical, histopathological, and molecular results a synergistic action in a second intention healing wound in sheep has been observed. Experimental wounds treated with cold plasma and MSCs showed a slower but more effective healing compared to the single treatment, as observed in previous studies. The combined treatment improved the correct development of skin appendages and structural proteins of the dermis showing the potential of the dual combination as a safe and effective tool for skin regeneration in the veterinary clinical field.

Autologous Platelet-Rich Plasma Enhances the Healing of Large Cutaneous Wounds in Dogs.

Platelet-rich plasma (PRP) is known to play a crucial role in skin wound healing, in both Human and Veterinary Medicine. Remarkably, until now, no studies have reported PRP treatment in subacute full-thickness skin wounds of the dog. The aim of this study was to evaluate the effects of two consecutive applications of autologous PRP, with the second application after 15 days, in 6 dogs showing large subacute skin wounds. The percentage of contraction, re-epithelialization and healing in all treated patients indicated that no complications or side effects, associated with consecutive PRP treatments, occurred in any patient and all wounds achieved complete closure and re-epithelialization. Our results suggest a positive effect of repeated autologous topical PRP treatments in large cutaneous subacute wounds of different etiology. Therefore, this PRP treatment could represent a simple, cost-effective, and valid alternative to promote healing processes in subacute large wounds cases in dogs.

Large Animal Models in Regenerative Medicine and Tissue Engineering: To Do or Not to Do.

Rapid developments in Regenerative Medicine and Tissue Engineering has witnessed an increasing drive toward clinical translation of breakthrough technologies. However, the progression of promising preclinical data to achieve successful clinical market authorisation remains a bottleneck. One hurdle for progress to the clinic is the transition from small animal research to advanced preclinical studies in large animals to test safety and efficacy of products. Notwithstanding this, to draw meaningful and reliable conclusions from animal experiments it is critical that the species and disease model of choice is relevant to answer the research question as well as the clinical problem. Selecting the most appropriate animal model requires in-depth knowledge of specific species and breeds to ascertain the adequacy of the model and outcome measures that closely mirror the clinical situation. Traditional reductionist approaches in animal experiments, which often do not sufficiently reflect the studied disease, are still the norm and can result in a disconnect in outcomes observed between animal studies and clinical trials. To address these concerns a reconsideration in approach will be required. This should include a stepwise approach using in vitro and ex vivo experiments as well as in silico modeling to minimize the need for in vivo studies for screening and early development studies, followed by large animal models which more closely resemble human disease. Naturally occurring, or spontaneous diseases in large animals remain a largely untapped resource, and given the similarities in pathophysiology to humans they not only allow for studying new treatment strategies but also disease etiology and prevention. Naturally occurring disease models, particularly for longer lived large animal species, allow for studying disorders at an age when the disease is most prevalent. As these diseases are usually also a concern in the chosen veterinary species they would be beneficiaries of newly developed therapies. Improved awareness of the progress in animal models is mutually beneficial for animals, researchers, human and veterinary patients. In this overview we describe advantages and disadvantages of various animal models including domesticated and companion animals used in regenerative medicine and tissue engineering to provide an informed choice of disease-relevant animal models.

From Food Waste to Innovative Biomaterial: Sea Urchin-Derived Collagen for Applications in Skin Regenerative Medicine.

Collagen-based skin-like scaffolds (CBSS) are promising alternatives to skin grafts to repair wounds and injuries. In this work, we propose that the common marine invertebrate sea urchin represents a promising and eco-friendly source of native collagen to develop innovative CBSS for skin injury treatment. Sea urchin food waste after gonad removal was here used to extract fibrillar glycosaminoglycan (GAG)-rich collagen to produce bilayer (2D + 3D) CBSS. Microstructure, mechanical stability, permeability to water and proteins, ability to exclude bacteria and act as scaffolding for fibroblasts were evaluated. Our data show that the thin and dense 2D collagen membrane strongly reduces water evaporation (less than 5% of water passes through the membrane after 7 days) and protein diffusion (less than 2% of BSA passes after 7 days), and acts as a barrier against bacterial infiltration (more than 99% of the different tested bacterial species is retained by the 2D collagen membrane up to 48 h), thus functionally mimicking the epidermal layer. The thick sponge-like 3D collagen scaffold, structurally and functionally resembling the dermal layer, is mechanically stable in wet conditions, biocompatible in vitro (seeded fibroblasts are viable and proliferate), and efficiently acts as a scaffold for fibroblast infiltration. Thus, thanks to their chemical and biological properties, CBSS derived from sea urchins might represent a promising, eco-friendly, and economically sustainable biomaterial for tissue regenerative medicine.

Parallels in the interactive effect of highly sensitive personality and social factors on behaviour problems in dogs and humans.

Sensory Processing Sensitivity (SPS) is a personality trait in humans characterised by a tendency to process information deeply, to be easily overstimulated, and to have strong emotional responses and an enhanced sensitivity to subtle stimuli. A trait similar to SPS has recently been identified in dogs ("canine Sensory Processing Sensitivity", cSPS). In children, this trait interacts with parenting factors to influence emotional and mental development, which in turn are linked to behaviour problems. Paralleling these findings in humans, we demonstrate that cSPS interacts with owner personality and use of aversive communication to influence the likelihood of behaviour problems in dogs. More behaviour problems were reported for more highly sensitive dogs per se, when there was a relative mismatch between owner and dog personality, and when use of "negative punishment" was reported. These findings indicate that a dog's personality might moderate how an individual is affected by environmental factors, particularly owner personality and communication style, emphasising the importance of considering individuality in prevention, development and treatment of behaviour problems in dogs.

Play like me: Similarity in playfulness promotes social play.

Social play is associated with the experience of positive emotions in higher vertebrates and may be used as a measure of animal welfare. Altering motivation to play (e.g., through short-term social isolation) can temporarily affect play levels between familiar individuals, a process which may involve emotional contagion. This study investigated how forming groups based on known differences in the personality trait "playfulness" (i.e., the longer-term propensity of an individual to actively play from adolescence to early adulthood) affects social play. Seventy-six adolescent male Lister Hooded rats underwent a Play-in-Pairs test assessing their playfulness, ranked as high (H), intermediate (I) or low (L). At seven weeks of age, rats were resorted into homogenous groups of similar (LLL, III, HHH), or heterogeneous groups of dissimilar (HII, LII) playfulness. Social play was scored in the home cage at Weeks 8, 10, 12 of age. A second Play-in-Pairs test was performed (Week 11) to assess consistency of playfulness. A Social Preference test investigated whether I rats in heterogeneous groups preferred proximity with I, H or L cage mates. It was found that heterogeneous groups played less than homogeneous ones at adolescence (8 weeks of age), while play levels at early adulthood (Weeks 10 and 12) did not differ between groups. Play in the homogeneous groups decreased with age as expected, while it did not change over time in the heterogeneous groups, which did not compensate for the lower play levels shown at adolescence. Play-in-Pairs scores before and after resorting were mildly correlated, indicating some level of consistency over time despite the resorting procedure. In the Social Preference test, subjects did not prefer one playfulness level over another. We conclude that a mismatch in playfulness may negatively affect social play development, and thus the welfare, of rats. Groups made of animals with similar playfulness, even those initially scoring relatively low in this trait, seemed to be more successful in establishing play relationships during adolescence.

Age-dependent variations in the expression of myosin isoforms and myogenic factors during the involution of the proximal sesamoidean ligament of sheep.

In ungulates the stability of the fetlock joint is dependent on several muscles, which are exposed to high stress and strain. Among those muscles, the proximal sesamoidean ligament or PSL (also known as the suspensory ligament or Ruini's elasto-tendinous organ) is organized at birth in layers of muscle fibres alternated with abundant tendinous tissue that, during the postnatal development, becomes the predominant tissue. In this study we analysed the PSL of the sheep at the age of 1, 30 and 180 days and determined the expression of several genes which either (a) are markers of muscle fibre growth and maturation, or (b) play a role as signal molecules. We observed an accelerated maturation, as indicated by the transition of MyHC isoform expression towards the slow isoforms and a reduced regenerative potential indicated by the low Pax7 expression and the altered Wnt signalling. We also found a specific myogenic expression pattern of MyoD, Myf5 and Myogenin in the developing PSL and high mRNA levels of specific fibrogenic factors, as TGF-ß1, that, undoubtedly, stimulate the growth of connective tissue. Our observations confirmed, at molecular level, the peculiarity of the fast involution observed in PSL a muscle that undergoes a very specific active differentiation process during early development, which implies myofibres involution and their replacement with connective tissue.

A cross-species judgement bias task: integrating active trial initiation into a spatial Go/No-go task.

Judgement bias tasks are promising tools to assess emotional valence in animals, however current designs are often time-consuming and lack aspects of validity. This study aimed to establish an improved design that addresses these issues and can be used across species. Horses, rats, and mice were trained on a spatial Go/No-go task where animals could initiate each trial. The location of an open goal-box, at either end of a row of five goal-boxes, signalled either reward (positive trial) or non-reward (negative trial). Animals first learned to approach the goal-box in positive trials (Go) and to re-initiate/not approach in negative trials (No-go). Animals were then tested for responses to ambiguous trials where goal-boxes at intermediate locations were opened. The Go:No-go response ratio was used as a measure of judgement bias. Most animals quickly learned the Go/No-go discrimination and performed trials at a high rate compared to previous studies. Subjects of all species reliably discriminated between reference cues and ambiguous cues, demonstrating a monotonic graded response across the different cue locations, with no evidence of learning about the outcome of ambiguous trials. This novel test protocol is an important step towards a practical task for comparative studies on judgement biases in animals.

Development of the "Highly Sensitive Dog" questionnaire to evaluate the personality dimension "Sensory Processing Sensitivity" in dogs.

In humans, the personality dimension 'sensory processing sensitivity (SPS)', also referred to as "high sensitivity", involves deeper processing of sensory information, which can be associated with physiological and behavioral overarousal. However, it has not been studied up to now whether this dimension also exists in other species. SPS can influence how people perceive the environment and how this affects them, thus a similar dimension in animals would be highly relevant with respect to animal welfare. We therefore explored whether SPS translates to dogs, one of the primary model species in personality research. A 32-item questionnaire to assess the "highly sensitive dog score" (HSD-s) was developed based on the "highly sensitive person" (HSP) questionnaire. A large-scale, international online survey was conducted, including the HSD questionnaire, as well as questions on fearfulness, neuroticism, "demographic" (e.g. dog sex, age, weight; age at adoption, etc.) and "human" factors (e.g. owner age, sex, profession, communication style, etc.), and the HSP questionnaire. Data were analyzed using linear mixed effect models with forward stepwise selection to test prediction of HSD-s by the above-mentioned factors, with country of residence and dog breed treated as random effects. A total of 3647 questionnaires were fully completed. HSD-, fearfulness, neuroticism and HSP-scores showed good internal consistencies, and HSD-s only moderately correlated with fearfulness and neuroticism scores, paralleling previous findings in humans. Intra- (N = 447) and inter-rater (N = 120) reliabilities were good. Demographic and human factors, including HSP score, explained only a small amount of the variance of HSD-s. A PCA analysis identified three subtraits of SPS, comparable to human findings. Overall, the measured personality dimension in dogs showed good internal consistency, partial independence from fearfulness and neuroticism, and good intra- and inter-rater reliability, indicating good construct validity of the HSD questionnaire. Human and demographic factors only marginally affected the HSD-s suggesting that, as hypothesized for human SPS, a genetic basis may underlie this dimension within the dog species.

Context-dependent individual differences in playfulness in male rats.

Play has been proposed as an indicator of positive emotions and welfare in higher vertebrates. This study investigated playfulness in male rats by exploring its consistency across motivational states (with/without prior short social isolation) and two age points at early and late adolescence. Twenty-four male Lister Hooded rats housed in cages of four underwent two play tests: conspecific Play-in-Pairs and Tickling by the experimenter, which were compared with play in the home cage and basal anxiety levels. Play-in-Pairs measures were consistent across age and motivational states, and were independent from anxiety. Positively valenced vocalizations in the Tickling test were also consistent across age, yet were negatively related to anxiety. Play-in-Pairs and Tickling play contexts, as well as social and solitary play types, were unrelated. Therefore, this study supports the existence of consistent individual differences in playfulness in rats, and suggests that different play contexts and types represent motivationally distinct systems.

A mini-review of TAT-MyoD fused proteins: state of the art and problems to solve.

The transcriptional activator TAT is a small peptide essential for viral replication and possesses the property of entering the cells from the extracellular milieu, acting as a membrane shuttle. In order to safely differentiate cells an innovative methodology, based on the fusion of transcription factors and the TAT sequence, is discussed in this short review. In several studies, it has been demonstrated that TAT protein can be observed in the cell nucleus after few hours from the inoculation although its way of action is not fully understood. However, further studies will be necessary to develop this methodology for clinical purposes.

Facial Indicators of Positive Emotions in Rats.

Until recently, research in animal welfare science has mainly focused on negative experiences like pain and suffering, often neglecting the importance of assessing and promoting positive experiences. In rodents, specific facial expressions have been found to occur in situations thought to induce negatively valenced emotional states (e.g., pain, aggression and fear), but none have yet been identified for positive states. Thus, this study aimed to investigate if facial expressions indicative of positive emotional state are exhibited in rats. Adolescent male Lister Hooded rats (Rattus norvegicus, N = 15) were individually subjected to a Positive and a mildly aversive Contrast Treatment over two consecutive days in order to induce contrasting emotional states and to detect differences in facial expression. The Positive Treatment consisted of playful manual tickling administered by the experimenter, while the Contrast Treatment consisted of exposure to a novel test room with intermittent bursts of white noise. The number of positive ultrasonic vocalisations was greater in the Positive Treatment compared to the Contrast Treatment, indicating the experience of differentially valenced states in the two treatments. The main findings were that Ear Colour became significantly pinker and Ear Angle was wider (ears more relaxed) in the Positive Treatment compared to the Contrast Treatment. All other quantitative and qualitative measures of facial expression, which included Eyeball height to width Ratio, Eyebrow height to width Ratio, Eyebrow Angle, visibility of the Nictitating Membrane, and the established Rat Grimace Scale, did not show differences between treatments. This study contributes to the exploration of positive emotional states, and thus good welfare, in rats as it identified the first facial indicators of positive emotions following a positive heterospecific play treatment. Furthermore, it provides improvements to the photography technique and image analysis for the detection of fine differences in facial expression, and also adds to the refinement of the tickling procedure.

Effect of Cage-Induced Stereotypies on Measures of Affective State and Recurrent Perseveration in CD-1 and C57BL/6 Mice.

Stereotypies are abnormal repetitive behaviour patterns that are highly prevalent in laboratory mice and are thought to reflect impaired welfare. Thus, they are associated with impaired behavioural inhibition and may also reflect negative affective states. However, in mice the relationship between stereotypies and behavioural inhibition is inconclusive, and reliable measures of affective valence are lacking. Here we used an exploration based task to assess cognitive bias as a measure of affective valence and a two-choice guessing task to assess recurrent perseveration as a measure of impaired behavioural inhibition to test mice with different forms and expression levels of stereotypic behaviour. We trained 44 CD-1 and 40 C57BL/6 female mice to discriminate between positively and negatively cued arms in a radial maze and tested their responses to previously inaccessible ambiguous arms. In CD-1 mice (i) mice with higher stereotypy levels displayed a negative cognitive bias and this was influenced by the form of stereotypy performed, (ii) negative cognitive bias was evident in back-flipping mice, and (iii) no such effect was found in mice displaying bar-mouthing or cage-top twirling. In C57BL/6 mice neither route-tracing nor bar-mouthing was associated with cognitive bias, indicating that in this strain these stereotypies may not reflect negative affective states. Conversely, while we found no relation of stereotypy to recurrent perseveration in CD-1 mice, C57BL/6 mice with higher levels of route-tracing, but not bar-mouthing, made more repetitive responses in the guessing task. Our findings confirm previous research indicating that the implications of stereotypies for animal welfare may strongly depend on the species and strain of animal as well as on the form and expression level of the stereotypy. Furthermore, they indicate that variation in stereotypic behaviour may represent an important source of variation in many animal experiments.

An Exploration Based Cognitive Bias Test for Mice: Effects of Handling Method and Stereotypic Behaviour.

Behavioural tests to assess affective states are widely used in human research and have recently been extended to animals. These tests assume that affective state influences cognitive processing, and that animals in a negative affective state interpret ambiguous information as expecting a negative outcome (displaying a negative cognitive bias). Most of these tests however, require long discrimination training. The aim of the study was to validate an exploration based cognitive bias test, using two different handling methods, as previous studies have shown that standard tail handling of mice increases physiological and behavioural measures of anxiety compared to cupped handling. Therefore, we hypothesised that tail handled mice would display a negative cognitive bias. We handled 28 female CD-1 mice for 16 weeks using either tail handling or cupped handling. The mice were then trained in an eight arm radial maze, where two adjacent arms predicted a positive outcome (darkness and food), while the two opposite arms predicted a negative outcome (no food, white noise and light). After six days of training, the mice were also given access to the four previously unavailable intermediate ambiguous arms of the radial maze and tested for cognitive bias. We were unable to validate this test, as mice from both handling groups displayed a similar pattern of exploration. Furthermore, we examined whether maze exploration is affected by the expression of stereotypic behaviour in the home cage. Mice with higher levels of stereotypic behaviour spent more time in positive arms and avoided ambiguous arms, displaying a negative cognitive bias. While this test needs further validation, our results indicate that it may allow the assessment of affective state in mice with minimal training-a major confound in current cognitive bias paradigms.